FIG. 6.

Cx32 inhibition decreased H24R4-induced ROS generation and distribution, and attenuated the activation of NF-κB/p53/PUMA-mediated mitochondrial apoptosis in HK-2 cells. Before H24R4 exposure, different methods were used to inhibit GJ function composed of Cx32 in HK-2 cells, including 2APB (gap junction inhibitor, 25μM, 1 h pretreatment), Gap27 (Cx32 peptide, 100μM, 24 h pretreatment), and specific Cx32-siRNA, to observe effects of Cx32 GJ function on HK-2 cellular ROS production, and expression alternation of mitochondrial apoptosis-related proteins. (A–C) Effects of 2APB, Gap27, and Cx32-siRNA on HK-2 cellular ROS production, detected with DHE staining (A, B, stained in red, scale bar 50 μm) and DCFH-DA staining (C). (D–G) Effects of Cx32-siRNA on expression of Cx32, p-p65, and p53 in HK-2 cells with Western blot. (H) Effects of Cx32-siRNA on expression of PUMA and caspase-3 with IF staining (PUMA, stained in green; caspase-3, stained in red; cell nuclei, stained in blue with DAPI, scale bar 50 μm). Data are presented as mean ± SE (n = 4). *p < 0.05 compared with control group; ^#p < 0.05 versus H24R4 group. ^$p < 0.05 compared with Cx32-NC+H24R4 group. DCFH-DA, 6-carboxy-2`-7`-dichlorodihydrofluorescein diacetate. Color images are available online.